A biofuel cell is an apparatus which can convert reducing equivalents generated from the energy metabolism of an organism into electric energy using the organism or a part of the organism. For generation of electricity using a microbial fuel cell, electrons generated from the energy metabolism of a microorganism should be transferred to an electrode. However, cells of all kinds of microorganisms are surrounded by a membrane, and such a membrane is made of nonconducting lipids. Thus, direct electron exchanges between a microorganism and an electrode cannot accomplished. Therefore, when a microorganism is used as a catalyst, a suitable mediator should be used to facilitate electron transfer between an organism and an electrode.
Roller et al. disclose a biofuel cell which utilizes as a catalyst Proteus vulgaris, Escherichia coli, Atcaligenes eutrophus, Azotobacter chroococum, Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas putida, etc. and as an electron transfer mediator thionine, methylene blue, brilliant cresyl blue, benzyl viologen, etc. [See, Roller et al., 1984, Journal of Chemical Technology and Biotechnology 34B:3-12]. Roller et al. confirmed that the efficiency of the biofuel cell compared to the amounts of the consumed oxygen varies depending on the bacteria used and the kind of the electron transfer mediator. Also, in a fuel cell in which glucose was used as a fuel, it appeared that the best bacteria in terms of the amount of oxygen consumption and the reduction rates of an electron transfer mediator were Escherichia coli and Proteus vulgaris [See, ibid].
In a biofuel cell using an electron transfer mediator, the rates of substrate consumption, the reduction rates of mediator by a microbe and the reactivity of an electron transfer mediator with electrodes, etc. are believed to be important factors for the efficiency of a biofuel cell. In addition, the metabolism of a microbe is influenced by the redox potential of an electron transfer mediator, thus the microbe's durability as a catalyst is also very important.
Robin et al. have constituted a biofuel cell with 45 mg of Proteus. vulgaris as a biocatalyst, 0.5 mM of hydroxynaphatoquinone (HNQ) as an electron transfer mediator, and 20 .mu.M of glucose as a fuel, which revealed an electromotive power of 0.5 milliamperes (mA) and 0.7 volts (V) [See, Robin et al., 1993, Applied Biochemistry and Biotechnology 39/40:27-40].
Bennetto et al. disclose a fuel cell constituted by sugar as a fuel, Proteus vulgaris as a catalyst and thionine as a mediator [See, Bennetto et al., 1985, Biotechnology Letters, 7:699-704]. This cell is reported to generate up to 44 coulombs (C) of electric current.
Meanwhile, Habermann and Pommer have reported that 150 mA per cm.sup.2 of electric current was generated from a fuel cell comprising as an electrode cobalt oxide, molybdenum/vanadium alloy in place of an electron transfer mediator, and as a fuel hydrogen sulfide which a sulfate reducing bacterium produces in waste water [See, Habermann and Pommer, 1991, Applied Microbiology and Biotechnology 33:128-133].
Biofuel cells developed hitherto essentially use an artificial electron transfer mediator or a mediator equivalent such as sulfate. Such an artificial mediator can promote the efficiency of a fuel cell. However, the artificial mediator has disadvantages that they should be used in a limited amount since it is toxic to the microorganisms, and exerts adverse effects on microbial populations. In addition, the artificial mediator may cause environmental problems when disposing of it after consumption. Even in a case where such an artificial mediator is not used, there has been a problem that a specific metal which is not eroded by hydrogen sulfide should be used as an electrode when constructing a fuel cell since the reductive metabolic products (i. e., hydrogen sulfide) produced by a microorganism should be used as an electron transfer medium.